液相色谱-质谱联用法测定人血浆中氯吡格雷及其3种代谢产物的浓度

Determination of Clopidogrel and Its Three Metabolites Concentrations in Human Plasma by LC-MS/MS

目的:建立同时测定人血浆中氯吡格雷(CLP)及其中间代谢产物2-氧-氯吡格雷(2-O-CLP)、非活性代谢产物氯吡格雷羧酸代谢物(CLPCA)和活性代谢产物氯吡格雷硫醇代谢物(CLPTM)浓度的方法。方法:选取陆军军医大学第一附属医院确诊为脑卒中的90名患者,早晨空腹口服1片氯吡格雷片(75 mg/片),于服药2 h后采集血样,将CLPTM经2-溴-3’-甲氧基苯乙酮衍生形成CLPTM-D后与其余3种待测物一起通过乙腈蛋白沉淀提取后,采用液相色谱-串联质谱法(LC-MS/MS)测定其浓度。色谱柱为Agilent poroshell 120 EC-C18,流动相为0.1%甲酸的乙腈溶液-0.1%甲酸水溶液(90∶10,V/V),采用多反应监测模式进行正离子检测,检测离子对分别为CLPCA质荷比(m/z)308.1→198.1、CLP m/z 322.3→212.0、2-O-CLP m/z 338.3→155.0、CLPTM-D m/z504.4→354.1、内标噻氯吡啶m/z 264.0→154.1。结果:CLPCA、CLP、2-O-CLP、CLPTM-D和内标的保留时间分别为2.01、3.32、2.83、2.68、1.87 min,CLPCA、CLP、2-O-CLP、CLPTM-D检测质量浓度的线性范围分别为100~10000、0.2~20、0.3~30、0.5~50ng/mL(r均≥0.9995),日内、日间精密度试验的RSD均≤9.5%(n=5),准确度为93.5%~98.9%(n=5),提取回收率为85.4%~95.9%(n=5),基质效应的CV为2.7%~6.2%(n=5)。稳定性(-80℃放置3个月、3次冷冻-解冻循环、4℃放置8 h)试验中,CLPCA、CLP、2-O-CLP和CLPTM-D的RE均≤10.0%(n=5)。结论:建立的LC-MS/MS法特异性强,测定结果准确可靠,可用于检测人血浆中CLP及其3种代谢产物的浓度。

OBJECTIVE:To establish the method for simultaneous determination of clopidogrel(CLP),its intermediate metabolite(2-O-CLP),inactive metabolite(CLPCA)and active metabolite(CLPTM)in human plasma.METHODS:Totally 90 patients diagnosed as stroke were selected from the First Affiliated Hospital of Army Medical University.They were given one CLP tablet(75 mg/tablet)orally on an empty stomach in the morning.Blood samples were collected 2 h after taking the tablet.CLPTMD was formed by derivation of CLPTM with 2-bromo-3’-methoxyacetophenone and extracted by precipitation of acetonitrile protein together with the other three substances to be measured.LC-MS/MS method was adopted.The determination was performed on Agilent poroshell 120 EC-C18 column with mobile phase consisted of acetonitrile(0.1%formic acid)and water(0.1%formic acid)(90∶10,V/V).The quantitation analysis was performed using multiple reaction monitoring at the specific ion transitions of m/z308.1→198.1(CLPCA),322.3→212.0(CLP),338.3→155.0(2-O-CLP),504.4→354.1(CLPTM-D)and 264.0→154.1(ticlopidine,internal standard),respectively.RESULTS:The retention time of CLPCA,CLP,2-O-CLP,CLPTM-D and internal standard were 2.01,3.32,2.83,2.68,1.87 min,respectively.The linear range of CLPCA,CLP,2-O-CLP and CLPTM-D were100-10000,0.2-20,0.3-30,0.5-50 ng/mL(all r≥0.9995).The intra-day and inter-day RSD were all less than 9.5%(n=5).Accuracy ranged from 93.5%-98.9%(n=5),and extraction recovery was from 85.4%to 95.9%(n=5).The matrix effect ranged from 2.7%-6.2%(n=5).In stability tests(storing at-80℃for 3 months,3 freeze-thaw cycles,storing at 4℃for 8 h),RE of CLP,CLPCA and CLPTM-D were all lower than 10.0%(n=5).CONCLUSIONS:Established LC-MS/MS method has the advantages of high specificity,accuracy and reliability,and can be used to detect the concentration of CLP and its three metabolites in human plasma.